Dehydro-sterol compounds and manufacture thereof



Patented Nov. 16, 1937 UNITED STATES DEHYDRO-STEBDL COlIPUNDS ANDMANUFAUIURE THEREOF Ado]! Windans and Friedrich Schenck, Gottingen,

Germany,

assignors to throp Chemical Company, Inc., New York, N. Y., acorporation of New York No Drawing. Application March 18, 1936, SerialNo. 69,590. In Germany March 20, 1935 13 Claims.

The Roman numerals indicate the usual numbering oi the tour rings andthe Arabic numerals CHI 45 Acy1--0 LOYI-J) (Cl. 2B0-153) indicate theusual numbering of the carbon atoms of the sterol ring system.

In accordance with the present invention the said '7-dehydrosteroicompounds are obtainable from well available sterols by starting withsterol 5 compounds containing in ring I! a keto group and a double bondin a, p-position thereto, or with esters of such sterol compounds,transforming the keto group by reduction with mild reducing agents intothe hydroxyl group while simultaneously maintaining the carbon doublebonds 0! the sterol compound, and then splitting ofl thehydroxyi groupin the form of water or, preferably, after steriflcation in the form 01an acid to produce a new double bond in ring II which stands inconjugated position to the other double bond originally present in ringI! of the sterol compound. when starting, for instance, with7-oxo-cholestero1 the production 01' 7-dehydrocholesterql'proceeds inaccordance with the iollowing reaction scheme:-

CHI

CHI CH:

H-CHr-CHrOHz-CH 0 reduction on; on. em

( iHCHaC r-CHr-GH on csta'iiicaiion heat decomposition 03' CH: lCH;H-CHr-Clir-CBr-GK saponiilcation 0111 CH- acid, is split off with theproduction of the dou- E, ble bond in ring II, whereas the acyl group7-Dehydrocholesterol (CuHnO).

are obtainable.

The keto derivatives of the sterols used as starting material may beobtained by oxidation of the esterifled sterols, for instance by meansof chromic acid in the manner known per se. Advantageously the esterderivatives of the keto sterols which are thus obtained are directlyused for the reduction with the mild reducing agent. As such preferablyan aluminium alcoholate is used, for instance aluminium methylate,ethylate, propylate and the like. Particularly suitable have proved thealcoholates of secondary alcohols, for instance aluminium isopropylate,isobutylate and isoamylate. Likewise the alcoholates of other earthmetals have proved to be suitable, furthermore, for instance magnesiumand zirconium alcoholates. Generally speaking metal alcoholates may beused which are capable of reducing aldehyde and keto groups to hydroxylgroupsbut do not develop free hydrogen in the reducing process. Thereduction is advantageously carried out in the presence of excessalcohol, preferably of that alcohol which is present in the metalalcoholate.

By removal of the hydroxyl group produced in the aforementionedreduction process, the second double bond standing in conjugatedposition to the originally present double bond is produced in ring II.The above reaction scheme shows the production of the second double bondin ring 11 by splitting ofi acid after previous esterification of thehydroxyl group. This esterification is preferably effected by means ofbenzoyl chloride, for instance in the presence of pyridine or quinoline.This procedure appears to yield better resultsthan the production of thesaid double bond by directly splitting off water from the non-esterifiedhydroxyl compound which, however, may also be performed advantageouslyin the presence of such agents as standing in the 3-pos'ition remainsunchanged. The 7-dehydro-3-acy1 compounds are transformed into the7-dehydro-sterols by saponification in the usual manner, for instance,by means of alkali in alcoholic solution. Caustic alkali andalkaline-earth metal hydroxides may be employed as the saponifyingagents.

The 'I-dehydro-sterols thus obtainable form white crystals. From theirsolution they may be;

precipitated by. digitonin in the usual manner. They show an absorptionspectrum which is similar to that of ergosterol. It is most surprisingthat when, for instance, subjecting 7-dehydrocholesterol to ultra-violetirradiation in.

the manner known per se an antirachitically active product is obtained.Accordingly, the new products render it possible to transform wellavailable sterol compounds which per se cannot be antirachiticallyactivated into antirachitically active products.

The invention is further illustrated by the fol- I examples withoutbeing restricted hours with a solution of 20 grams of chromic acidanhydride in 900 cos. of glacial acetic acid.

The oxidation solution is kept at a temperature of 50 C. for 9 hours.The acetic acid solution which has turned green is concentrated underreduced pressure, diluted with water and extracted with ether. The etherextract is freed from acid constituents by washing with dilute therpurification the product is recrystallized from hot methanol. The7-oxo-stigmasterol acetate obtained in this manner melts at 183 C. I

The 7-hydroxystigmasterol is obtained by dissolving 10 grams of'l-oxo-stigmasterol acetate in 200 cos. of isopropyl alcohol, treatingthe solution with 20 grams of liquefied aluminium-isopropylate andboiling for 4 hours under reflux. After the reduction is complete theyellow, greenfluorescent solution is diluted with ether and freed fromaluminium and isopropyl alcohol by washing with dilute hydrochloricacid. The colorless ether extract is evaporated after drying and theresidue taken up in a small quantity of methanol. After several hoursthe 7-hydroxystigmasterol has separated in tufts of thin needles whichare recrystallized from methanol. The 7-hydroxy-stigmasterol melts at154 C. On heating with chloral hydrate a deep blue melt is obtainedafter a few seconds. Reactions according to Salkowski: sulfuric acidred. chloroform blue, intensive green fluorescence.

3.5 grams of 7-hydroxy-stigmasterol are dissolved in 200 005. ofpyridine and treated with a solution of 5 cos. of benzoyl chloride in 10cos. of pyridine. The mixture is heated for 30 minutes on thewater-bath, diluted after cooling with ether, extracted with water anddilute hydrochloric acid for removing the pyridine. The dried etherextract is concentrated to a small volume and treated with methanoluntil turbidity s multaneously.

occurs. If the dibenzoate should separate as a jelly, ether is addeduntil solution is complete. After standing for a prolonged time thedibenzoate separates in thin woolly needles. It is purified byrecrystallization from ether-methanol. The7-hydroxy-stigmasterol-dibenzoate melts at 154-158 C. and displays thesame color reactions as the 7-hydrom-stigmasterol.

2.13 grams of 7-hydroxy-stigmasterol-dibenmate are heated in a water-letvacuum (1042 mm. pressure) to 190-200 C. until about 0.3 gram, that is60% of the theoretical quantity of benzoic acid (theoretically 0.40gram) are split oil. -The melt is dissolved in a small quantity of etherafter cooling. After standing for a short time the monobenzoate of the'l-dehydrostigmasterol sep-.

arates from the ethereal solution in thin needles. For furtherpurification the solution is recrystallized from ether-methanol. The7-dehydrostigmasterol-benzoate melts at 178480" C. It 'is' dimcultlysoluble in methanol, slightly soluble in ether and acetone. Furtherquantities of monobenzoate may be obtained from the mother lyes byheating again and working up in the above described manner.

7-dehydrostigmasterol-benzoate in benzene. The

mixture is heated to boiling and the solvent distilled of! after boilingfor half an hour until crystallization occurs. The crude product isrecrystallized from ether-methanol for purification.'Z-dehydrostigmasterol crystallizes in long, thin needles. and melts at153 C. It is dimcultly soluble in methyland ethyl-alcohol, more readilysoluble in ether or acetone and displayswith antimony trichloride thecolor reacton of ergosterol (red-blue), yields the ergosterol spectrumand can be precipitated with digitonin. 7

Example 2.-For obtaining 'I-hydroxy-cholesterol 50 grams of'I-oxo-cholesteryl acetate are dissolved in 500 ccs. of dry isopropylalcohol and after the addition of 20 grams of liquefied alu-.

minium isopropylate heated to boiling for 5 hours.

After reduction is complete with the simultaneous splitting ofi of theacetyl group the isopropylalcoholic solution is diluted with ether. Theethereal solution is first extracted with normal hydrochloric acid,then, after removing the aluminium hydroxide, with water. The driedethereal solution is concentrated to about -150 ccs. and treated withthe five-fold quantity of petroleum ether, whereupon abundant quantitiesof the l-hydroxy cholesterol separate in colorless jelly balls which arefiltered with suction and washed with petroleum ether. By evaporatingthe mother liquor to a small volume and by diluting with petroleum etherfurther quantities of 7-hydroxy-cholesterol may be obtained. It yieldsimmediately an intensive blue coloration when heated on the water bathwith chloral hydrate. In the Salkowski reaction the sulfuric acid takesa red coloration, the chloroform a blue coloration, an intensive greenfluorescence occurs With antimony trichloride in chloroform a deep bluecoloration is obtained after a short time.

For obtaining '1-hydroxycholesterol-dibenz0ate 30 grams of7-hydroxy-cholesterol are dissolved in 200 cos. of pyridine and treatedwith a mixture of 30 grams of benzoyl chloride and 30 grams of pyridine.After standing for 24 hours the benzoylation product is precipitatedwith 600 cos. of water. The oily precipitate after settling. is

again shaken with 500 cos. of fresh water. The

water is poured off and the crude benzoate, covered with methyl alcohol,is left standing for several hours, whereupon it gradually turns to awhite crystal powder. The latter is filtered, washed with methyl alcoholand recrystallized from ether-methyl alcohol for further purification.In this manner thin needles are obtained in a good yield which melt at1'10 C. The '7- hydroxy-cholesteroi-dibenzoate displays the same colorreactions as the 7-hydroxy-cholesterol.

For transforming into the 'I-dehydrocholesterol-benzoate the7-hydroxycholesterol-dibenzoate is heated in portions 01 2 grams eachfor one hour at 19u-200 C. at about 1 mm. pressure. Besides benzoic acidsmall quantities of oily constituentsdistil over which are removedbefore further working up. The glass-like mass re maining afterheatingabout 60% of the dibenzoate employed-is dissolved with ether oracetone and the solution concentrated to a small volume. After standingfor a short time the monoben zoate of the 7-dehydrocholesterol separatesin crystals. The crude monobenzoate is purified by recrystallizationfrom ether or acetone. In this manner thin leaflets are obtained whichyield a turbid melt at 142-143 C. which becomes clear at C. The productdisplays the same color reactions as the non-esterified7-hydroxycholesterol.

For transforming into the 7-dehydrocholesterol 10 grams of7-dehydrocholesterol-benzoate are dissolved in 50 cos. of benzene andtreated with 200 cos. of a 5% ethyl-alcoholic caustic potash solution byheating to boiling. After boiling for half an hour under refiux thesolvent is distilled of! eventually with subsequent addition of ethylalcohol until the benzene is removed and crystallization commences. The'I-dehydrocholesterol is filtered ofi after cooling and purified byrecrystallization from ether-methyl-alcohol. The 'Z-dehydrocholesterolwhich is obtained in almost theoretical yield" crystallizes in thinleaflets, melts at 149-150 C., may be precipitated with digitonln andhas an absorption spectrum which resembles It is difllcultly soluble inmethyl alcohol, readil soluble in ether and displays with antimonytrichloride in chloroform the color reaction of the ergosterol(red-blue) The dehydro-cholesterol may also directly be obtained from7-hydroxycholesterol by heating the latter compound with anhydrousoxalic acid or benzoic acid anhydride for some time in nitrogenatmosphere to a higher temperature, for instance, ISO-190 C. A brownishcolored melt is obtained which is dissolved in ether, diluted withmethanol, treated with a quantity of 10% methyl-alcoholic caustic potashsolution which is equivalent to the quantity of oxalic acid or benzoicacid anhydride employed, for saponifying the ester eventually formed,the ether and the greatest part of the methyl alcohol are then distilledoff. On cooling of the concentrated methyl-alcoholic solution thedehydro-cholesterol separates in crystals.

Example 3.For obtaining the 7-hydroxysitosterol 100 grams of sitosterolacetate are dissolved in 5 liters of hot glacial acetic acid, treatedafter cooling to 50-60" C. with a solution of 90 grams of chromic acidanhydride in glacial acetic acid and kept at a temperature of 50-60 C.for 5 hours. The glacial acetic acid solution is concentrated to about300 ccs. under reduced pressure, the residue is extracted with ether andthe ether extract freed from acid constituents by extraction with sodiumcarbonate solution. The ether extract is concentrated to a small volumeand treated with methanol until crystallization commences. The crudeproduct is recrystallized from ethermethanol. The 'l-oxo-sitosterolacetate melts at 154 0., crystallizes in'leaflets, is readily soluble inether and acetone and diflicultly soluble in methyl alcohol.

For transforming into the 'I-hydroxy-sitosterol 25 grams of'l-oxo-sitosterol acetate are dissolved in 250 cos. of dry isopropylalcohol, 10 grams of aluminium-isopropylate are added and the mixtureheated to boiling for 5 hours under reflux. After reduction is completewith simultaneous splitting oilf of the acetyl group the solution ispoured into about 3 liters of 0.5% aqueous caustic soda solution. Thereduction product which sep- 7-hydroxy-sitosterol separates in the formofsmall jelly balls.

For obtaining the 'l-hydroxy-sitosterol-dibenzoate 20 grams of7.-hydroxy'-sitosterol are dissolved in cos. of pyridine, treated with25 cos. of benzoyl chloride and left standing for 15 hours at roomtemperature. The pyridine solution is diluted with ether-water, theether extract is freed from pyridine by means of dilute hydrochloricacid, washed until free from acid, dried over sodium sulfate andconcentrated to about 50 cos. on treating the ethereal solution withmethanol until turbidity occurs, the dibenzoate separates in thinneedles. The 'I-hydroxy-sitosterol-dibenzoate melts afterrecrystallization from ethermethanol or acetone-methanol at C.

For transformation into the 'l-dehydro-sitosterol-benzoate the7-hydroxy-sitosterol-dibenzoate is heated in portions of 1 gram each inhigh vacuo for half an hour. Thereupon benzoic acid and oilyconstituents separate which before further worldng up are dissolved andremoved. The residue obtained after heating is dissolved in ether, theethereal solution is concentrated to a small volume and treated withmethanol until turbidity occurs. The monobenzoate which separates isrecrystallized from ether-methyl alcohol for further purification. The"I-dehydro-sItosterol-benzoate crystallizes in needles and melts at144-146" C.

For obtaining the 'l-dehydro-sitosterol 1 gram of'I-dehydro-sitosterol-benmate is dissolved in 10 cos. of hot benzene andtreated with 30 cos. of 5% sodium methylate solution by heating toboiling. The solvent is then distilled off until crystallizationcommences. For further purification the solution is recrystallized fromether-methyl alcohol. The 7-dehydro-sitosterol crystallizes in largeleaflets. It melts at 148-150" C. and is readily soluble in ether,diillcultly soluble in methylor ethyl alcohol. It may be precipitatedwith digitonin. On treating its chloroform solution with antimonytrichloride red coloration immediately takes place which after somestanding turns to blue. The absorption spectrum is similar to that ofergosterol.

We claim:--

1. Dehydrosterol compounds selected from the group consisting of7-dehydrocholesterol, 7-dehydrositosterol and 7-dehydrostigmasterol.

2. "l-dehydrocholesterol, having in the crystalacid with the productionof a 'l-dehydro compound and saponifying the 7-dehydro-monoestercompound.

4. The process which comprises reacting upon an esterifled sterolcompound containing in ring II a keto group and a double bond in a,p-position thereto with an aluminium alcoholate to reduce the 'l-ketogroup to the l-hydroizyl group, esterifying the hydroxyl groups of thereduction product, heating the diacyl compound formed to split of! theacyl radical in the 'T-position in the form of acid with the productionof a 7-dehydro compound and saponifying the 7-dehydromonoester compound.

5. The process which comprises reacting upon an acetylated sterolcompound containing in ring I! a keto group and a double bond in a,p-position thereto with an alcoholate of a metal selected from the groupconsisting of earth metals, zirconium and magnesium to reduce the'l-keto group to the 'l-hydroxyl group, esterifying the hydroxyl groupsof the reduction product, heating the diacyl compound formed to splitoil the acyl radical in the 7-position in the form of acid with theproduction of a 'I-dehydro compound and saponifying the7-dehydro-monoester com- Pound.

Y 6. The process which comprises reacting upon an esterifled sterolcompound containing in ring II a keto group and a double bond in a,B-p0sition thereto with an alcoholate of a metal selected from the groupconsisting of earth metals,

the benzoyl radical in the 'l-position in the form of benzoic acid withthe production of a 'l-dehydro compound and saponifying theY-dehydromonobenzoyl compound. I

'7. The process which comprises reactingupon an esterlfled sterolcompound containing in ring 11 a keto group and a double bond in a,p-position thereto with an aluminium alcoholate to reduce the 'l-ketogroup to the 'i-hydroxyl group, benzoylating the hydroxyl groups of thereduction product, heating the dibenzoyl compound formed to split oi thebenzoyl radical in the '7- position in the form of benzoic acid with theproduction of a 'l-dehydro compound and saponifying the7-dehydro-monobenzoy1 compound.

8. The process which comprises reacting upon an acetylated sterolcompound containing in ring 11 a keto group and a double bond in a,B-position thereto with an aluminium alcoholate to reduce the 'l-ketogroup to the 'I-hydroxyl group, benzoylating the hydroxyl groups of thereduction product; heating the dibenzoyl compound formed to split offthe benzoyl radical in the 7- position in the form of benzoic acid withthe production of a 'I-dehydro compound and saponifying the7-dehydro-monobenzoyl compound.

a '7-oxo-cholesterol ester with an alcoholate of a metal selected fromthe group consisting of earth metals, zirconium and magnesium to reducethe "I-keto group to the 'I-hydroxyl group, esterifyingthe hydroxylgroups of the reduction product, heating the diacyl compound formed tosplit oil? the acyl radical in the 7-posltion in the form 01' acid withthe production of a. 'I-dehydro cholesterol-ester and saponifying thelatter compound.

10. The process which comprises reacting upon a 7-oxo-cholesterol esterwith an aluminium alcoholate to reduce the '1-keto group to the'l-hydroxyl group, esterifyin'g the hydroxyl groups of the reductionproduct, heating the diacyl compound formed to split oi! the ac-ylradical in the 7-position in the form of acid with the production of a'I-dehydro cholesterolester and saponifying the latter compound.

11. The process which comprises reacting upon a 7-oxo-cholesterol esterwith an alcoholate of a. metal selected from the group consisting offormed to split oi the benzoyl radical in the '1- positlon in the formof benzoic acid with the production of 'I-dehydro cholesterol-benzoateand saponifying the latter compound.

12. The process which comprises reacting upon 7-oxo-cholesterol acetatewith an aluminium alcoholate to reduce the 'I-oxo-group to the7-hydroxyl group, benzoylating the hydroxyl groups, 'of the reductionproduct, heating the dibenzoate formed to split oi! the benzoyl radicalin the 7-position in the form of benzolc acid with the formation of7-dehydrocholesterol-monobenzo- -monobenzoate and saponifying the lattercomearth metals, zirconium and magnesium to repound. duce the '7-ketogroup to the 7-hydroxyl group, ADOLF WINDAUS. benzoylating the hydroxylgroups of the reduc- FRIEDRICH ,SCHENCK. tion product, heating thedibenzoyl compound Certificate of Correction Patent No. 2,098,984.

ADOLF WINDAUS ET AL.

7 November 16, 1.937.

It is hereby certified that error appears in the printed specificationof the above numbered patent requiring COITBOHOII'BS follows: Page 1,first column, lines 11 to 18,

inclusive, for the formula and that the said Letters Patent should beread with this correction therein that the same may conform to therecord ofthe case in the Patent Ofiice.

. Signed and sealed this 1st day of March, A. D. 1938.

HENRY VAN ABSDALE, Act'mg Commissioner of Patents.

Disclaimer 2,098,984.-Adolf Window and Ffied rich Sclmwk, Gottingen,Germany,

Dmimn Tmmo O-BTEBOL Conrotm'oe m MANUIAOIURI 1'. Patent a'7-oxo-cholesterol ester with an alcoholate of a metal selected from thegroup consisting of earth metals, zirconium and magnesium to reduce the"I-keto group to the 'I-hydroxyl group, esterifyingthe hydroxyl groupsof the reduction product, heating the diacyl compound formed to splitoil? the acyl radical in the 7-posltion in the form 01' acid with theproduction of a. 'I-dehydro cholesterol-ester and saponifying the lattercompound.

10. The process which comprises reacting upon a 7-oxo-cholesterol esterwith an aluminium alcoholate to reduce the '1-keto group to the'l-hydroxyl group, esterifyin'g the hydroxyl groups of the reductionproduct, heating the diacyl compound formed to split oi! the ac-ylradical in the 7-position in the form of acid with the production of a'I-dehydro cholesterolester and saponifying the latter compound.

11. The process which comprises reacting upon a 7-oxo-cholesterol esterwith an alcoholate of a. metal selected from the group consisting offormed to split oi the benzoyl radical in the '1- positlon in the formof benzoic acid with the production of 'I-dehydro cholesterol-benzoateand saponifying the latter compound.

12. The process which comprises reacting upon 7-oxo-cholesterol acetatewith an aluminium alcoholate to reduce the 'I-oxo-group to the7-hydroxyl group, benzoylating the hydroxyl groups, 'of the reductionproduct, heating the dibenzoate formed to split oi! the benzoyl radicalin the 7-position in the form of benzolc acid with the formation of7-dehydrocholesterol-monobenzo- -monobenzoate and saponifying the lattercomearth metals, zirconium and magnesium to repound. duce the '7-ketogroup to the 7-hydroxyl group, ADOLF WINDAUS. benzoylating the hydroxylgroups of the reduc- FRIEDRICH ,SCHENCK. tion product, heating thedibenzoyl compound Certificate of Correction Patent No. 2,098,984.

ADOLF WINDAUS ET AL.

7 November 16, 1.937.

It is hereby certified that error appears in the printed specificationof the above numbered patent requiring COITBOHOII'BS follows: Page 1,first column, lines 11 to 18,

inclusive, for the formula and that the said Letters Patent should beread with this correction therein that the same may conform to therecord ofthe case in the Patent Ofiice.

. Signed and sealed this 1st day of March, A. D. 1938.

HENRY VAN ABSDALE, Act'mg Commissioner of Patents.

Disclaimer 2,098,984.-Adolf Window and Ffied rich Sclmwk, Gottingen,Germany,

Dmimn Tmmo O-BTEBOL Conrotm'oe m MANUIAOIURI 1'. Patent

